Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.     

Mitochondrial membrane potential in cardiac myocytes

Mitochondria are involved in cellular functions that transcend the traditional role of these organelles as the energy factory of the cell. Their relative inaccessibility and the difficulties involved in attempts to study them in their natural environment -- the cytosol -- has delayed much of this understanding and they still have many secrets to yield. One of the relatively new fields in this respect is undoubtedly the analysis of mitochondrial membrane potential. The realization that its alteration may have important pathophysiological consequences has led to an increased interest in measuring this variable in a variety of biological settings, including cardiovascular diseases. Measurements of mitochondrial membrane potential tell us much about the role of mitochondria in normal cell function and in processes leading to cell death. However, we must be aware of the limitations of using isolated mitochondria, single cells and different fluorescent indicators.     

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

In these experiments we have investigated the feasibility and accuracy of recording steady-state and dynamic changes in transmembrane potential noninvasively across an intact cell-attached patch using the current-clamp mode of a conventional patch-clamp amplifier. Using an equivalent circuit mimicking simultaneous whole-cell voltage-clamp and cell-attached current-clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole-cell potential recorded across an intact membrane patch. This analysis revealed a steep increase in the accuracy of recording of steady-state membrane potential as the seal/membrane ratio increases from 0. The recording accuracy approaches 100% as the seal/membrane ratio approaches infinity. Membrane potential measurements across intact cell-attached patches in rat basophilic leukemia cells and rat megakaryocytes revealed a surprisingly high degree of accuracy and demonstrated the ability of this noninvasive technique to follow dynamic changes in potential in nonexcitable cells.     

Caffeine-sensitive calcium stores in presynaptic nerve endings: a physiological role?

Ca2+-sensitive minielectrodes and the fluorescent cytosolic calcium probes, quin2 and fura2, were used to study some aspects of calcium homeostasis in intact and permeabilized synaptosomes from whole rat brain. Experiments in permeabilized synaptosomes revealed the existence of a vesicular, non-mitochondrial, ATP-dependent calcium uptake system with a vanadate sensitivity similar to that of brain microsomes, or endoplasmic reticulum-type calcium sequestering organelles. By using the fluorescent probes it was possible to show that caffeine mobilizes calcium from these internal stores in intact synaptosomes. Our results indicate a role of the caffeine sensitive calcium stores in the buffering of calcium loads elicited by plasma membrane depolarization.     

The mitochondrial network of human neutrophils: role in chemotaxis,phagocytosis, respiratory burst activation,and commitment to apoptosis

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.     

A real-time view of life within 100 nm of the plasma membrane

The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.     

Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii

Two different approaches to prepare and characterise vacuoles from the filamentous fungus Ashbya gossypii are described, i.e. the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane. By mechanical lysis of protoplasts and separation of the organelles on a stepped density gradient, we obtained a vacuolar fraction virtually free of contamination by other organelles, unlysed protoplasts and cell debris. The integrity of the isolated organelles was characterised by vital-staining, the presence of α-mannosidase, and retained accumulation of basic amino acids. In a second approach, the cell membrane of the fungus was selectively permeabilised by use of the saponin digitonin leaving the vacuoles in their physiological surrounding, i.e. protected by the rigid cell wall. The permeabilisation was monitored by the latency of predominantly cytosolic amino acids and the ATP status of the cells. Functional intactness of the vacuoles within the permeabilised hyphae was demonstrated by maintenance of the pH gradient across the vacuolar membrane as detected by accumulation of the fluorescent dye, Acridine orange. These two methods are well-suited tools for the in situ assay of intracellular compartmentation of metabolites, for vacuolar transmembrane fluxes in Ashbya gossypii, as well as for the direct access to vacuolar membranes and enzymes of this fungus.     

Changes in secondary structure of poliovirus ribonucleic acid.

The incorporation of the fluorescent amine, dansyl cadaverine [N(5-aminopentyl)-5-dimethylamino-1-naphthalene sulfonamide], into the plasma membranes of intact cells was investigated. Using a fluorescent microscope, incorporation was observed when cultured mouse lymphoma (L1210) cells, cultured human fibroblasts and white cells from several sources were incubated in the presence of 0.1 mM dansyl cadaverine. While intact erythrocytes from several species did not incorporate the fluorescent amine, erythrocyte ghosts did. The uptake of dansyl cadaverine by L1210 cells was dependent upon the cell concentration, incubation time and temperature. Experiments designed to elucidate the structural requirements for fluorophor uptake demonstrated that, in addition to a hydrophobic dansyl group an extended straight hydrocarbon side chain with either an amino or hydroxyl group was necessary. The incorporated fluorophor was noncovalently associated with the cell membrane as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes and extraction of dansyl cadaverine labelled cells with choroform/methanol (2:1). These results indicate that dansyl cadaverine is incorporated into plasma membranes and suggest its potential usefulness as a new fluorescent probe in cell membrane studies.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Fluorescence of neurotransmitters and their reception in plant cell

The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.     

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.     

Early plasma membrane depolarization by alpha interferon: biologic correlation with antiproliferative signal

Daudi lymphoma cells, of a line sensitive to growth inhibition by alpha interferon, showed dose-dependent plasma membrane depolarization within 10 min after exposure to natural or recombinant alpha interferons (10 to 1000 IU/ml). This biophysical change was detected flow cytometrically by measuring the intensity of fluorescent emission from cells stained with dye indicators of membrane potential. Subclones of Daudi lymphoma cells, resistant to growth inhibition by alpha interferon, showed no membrane depolarization. Parallel results were obtained in initial tests of an isologous pair of T cell and B cell lines which differ in sensitivity to growth inhibition. Thus, decreased membrane potential may herald an interferon signal for antiproliferative action.     

Membrane topography of cardiac triadin.

Fusion constructs of partial sequences of triadin that contain green fluorescent protein at the N-terminus and glutathione transferase at the C-terminus have been expressed in human embryonic kidney -293 cells. A comparison of the subcellular disposition of a range of triadin fusion peptides indicates localization either to a few large organelles as a default target or to endoplasmic reticulum when amino acids 68-98 are present and structurally intact. Fluorescence from the conjugate of monochlorobimane with glutathione identifies whether the C-terminus has a cytoplasmic or luminal location. A stable transit of the membrane occurs in triadin2-98. Triadin2-117 and 2-267 give both cytoplasmic and luminal C-termini. Both triadin89-117 and triadin89-267 distribute in membranes, but do not cross them. The data are interpreted to indicate that cardiac triadin contains an alpha-helical membrane transit through the hydrophobic domain, 49-68, and a membrane association through the short hydrophobic domain, 102-114.     

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.     

Microdosimetry for conventional and supra-electroporation in cells with organelles

Conventional electroporation (EP) by 0.1 to 1 kV/cm pulses longer than 100 micros, and supra-electroporation by 10 to 300 kV/cm pulses shorter than 1 micros cause different cellular effects. Conventional EP delivers DNA, proteins, small drugs, and fluorescent indicators across the plasma membrane (PM) and causes moderate levels of phosphatidylserine (PS) translocation at the PM. We hypothesize that supra-EP is central to intracellular effects such as apoptosis induction and higher levels of PS translocation. Our cell system model has 20,000 interconnected local models for small areas of the PM and organelle membranes, small regions of aqueous media, appropriate resting potentials, and the asymptotic EP model. Conventional EP primarily affects the PM, but with a hint of endoplasmic reticulum involvement. Supra-EP can involve all of a cell's membrane at the largest fields. Conventional EP fields tend to go around cells, but supra-EP fields go through cells, extensively penetrating organelles.     

Flux measurement in single cells by fluorescence microphotolysis

Fluorescence microphotolysis — widely employed for diffusion studies — can be used to measure transfer (flux) of fluorescent solutes through membranes in single cells and organelles. This article analyses the methodological basis of flux measurements, provides experimental tests, and discusses potential applications. The principle of the method is to equilibrate cells, organelles or vesicles with a fluorescent solute, to deplete the interior of individual cells etc. of fluorescene by the pulse of a high-intensity microbeam, and to monitor influx of solute by microfluorometry. Simple equations are given and a computer curve fitting program is described by which rate constants of influx and membrane permeability coefficients can be derived from fluorescence measurements. The permeability of individual leaky human erythrocyte ghosts to fluorescein-isothiocyanate-labelled bovine serum albumin has been measured under various conditions. Multiple exposure to the high-intensity microbeam had no effect on permeability within experimental error. Flux measurements have been also performed on individual vesicles of 1–2 m radius which had been derived from ghosts. The potential application of the method to sub-lightmicroscopic vesicles and to organelles within living cells is discussed.Abbreviation FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin     

Changes in surface area of intact guard cells are correlated with membrane internalization

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.     

Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes

When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.     

Regulation of mitochondrial morphology by membrane potential,and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.